Delivering proteins to replace malfunctioning ones or to direct or regulate normal biological functions holds great promises for a broad spectrum of applications ranging from therapeutics, tissue engineering to other areas (see, e.g. D. E. Golan, et al. Nat. Rev. Drug Discovery 2008, 7, 21). However, while various delivery approaches have been established, developing the capability to deliver proteins according to local environmental changes, e.g., pH (see, e.g. D. J. Irvine, et al. Nano Lett. 2007, 7, 3056; D. J. Irvine, et al. Biomacromolecules 2009, 10, 756), and glucose (see, e.g. K. Kataoka, et al. J. Am. Chem. Soc. 1998, 120, 12694) and enzyme concentrations (see, e.g. K. S. Anseth, et al. Biomaterials 2009, 30, 6048; O. Franssen, et al. J. Controlled Release 1999, 59, 219) remains highly challenging.
It is known that biological systems often secrete specific enzymes in response to certain cellular events such as injury or disease (see, e.g. J. S. Powell, et al. Science 1989, 245, 186; L. L. Ji, Med. Sci. Sport Exercise 1993, 25, 225). If such enzymes could be used to trigger a polypeptide delivery system to release its protein cargoes, proteins could be delivered based on specific cellular events or environmental changes. Moreover, because enzymes are generally highly specific and secreted with precise spatial and temporal control; such an enzyme-responsive delivery can allow protein delivery with spatial and temporal control, factors which provide huge opportunities for tissue engineering and other applications.
The ability to control the delivery of agents into selected physiological environments is desirable. For example, growth factors (GF) such as vascular endothelial growth factor are widely used in tissue engineering applications to induce and guide blood vessel formation. However, the incorporation of GFs into matrices such as hydrogel scaffolds typically results in a loss of GF activity due to reactions between the GF and the reactive hydrogel precursors. For example, the introduction of GF to Michael addition crosslinked hydrogels that use dithiol-containing peptides for crosslinking results in a reduction of disulfide bridges in the encapsulated GF and reduced protein activity (see, e.g. Lutolf, M. P. et al. Nat Biotechnol 2003, 21, 513-518; Lutolf, M. P. & Hubbell, J. A. Biomacromolecules 2003, 4, 713-722; Lutolf, M. P., et al. Bioconjug Chem 2001, 12, 1051-1056; Zisch, A. H. et al. Faseb J 2003, 17, 2260-2262; Elbert, D. L. & Hubbell, J. A. Biomacromolecules 2001, 2, 430-441; Elbert, D. L., et al. J Control Release 2001, 76, 11-25).
Conventional technologies for protein delivery from matrices such as hydrogel scaffolds rely mainly on attaching protein-containing polymer microspheres to hydrogel scaffolds or directly immobilizing protein covalently or electrostatically to hydrogel scaffolds (see, e.g. A. M. Gopferich, et a. Adv. Drug Delivery Rev. 2007, 59, 274; K. Ladewig, Expert Opin. Drug Delivery 2011). The use of enzymatic action for protein delivery is limited to bulk hydrogels containing enzyme-responsive linkers, which can be cleaved off by specific enzymes to release their protein cargos (see, e.g. K. Ladewig, Expert Opin. Drug Delivery 2011; J. L. West, et al. Biomaterials 2010, 31, 3840; A. J. Garcia, et al. Proc. Natl. Acad. Sci. USA 2010, 107, 3323; A. H. Zisch, et al. FASEB J. 2003, 17, 2260). Nano-hydrogels have been synthesized using micro-emulsion polymerization for the general purpose of drug delivery (see, e.g. R. Kopelman, et al. Angew. Chem. Int. Ed. 2007, 46, 2224; R. Kopelman, et al. Nano Lett. 2008, 8, 3320). However, the synthesis of such nano-hydrogels has been generally achieved in reaction media containing a significant amount of organic solvent and surfactant, forbidding effective incorporation of active proteins within such nano-hydrogels.
There is a general need for methods and materials that can protect polypeptides such as proteins from certain environments, while simultaneously allowing them to function in other environments. The invention disclosed herein addresses these and other needs while overcoming many of the drawbacks and disadvantages of conventional methodologies.